In general, the salts of active pharmaceutical ingredients containing an amino group, which are commercially available or under medical investigation activity, with the exception of the quaternary ammonium salts, are generally prepared by contacting the corresponding amine with an organic or inorganic acid and are crystallized from solvents; see, for instance, the preparation of the following methanesulfonate salts (mesylates): amidephrine mesylate (RN 1421-68-7), betahistine mesylate, bromocriptine monomesylate (25614-03-3), deferoxamine mesylate, dihydroergocristine, dihydroergotamine mesylate, doxazin mesylate, perfloxacin monomesylate dehydrate (RN 70458-95-6), nelfinavir mesylate (RN 159989-65-8), pergolide mesylate (RN 66104-23-2), phentolamine mesylate, and saquinavir monomesylate (RN 149845-06-7) and the preparation of other salts of API such as indinavir sulphate (RN 157810-81-6), omoconazole nitrate (RN 83621-06-1), quinine sulphate (RN 549-56-4), racefemine hydrogen fumarate (RN 1590-35-8), ramosetron hydrochloride (RN 132907-72-3), and ropivacaine hydrochloride (RN 98717-15-8).
The crystallization of the salts has the aim to increase the salt purity by removing impurities which can be classified (Duane A. Pierson et al., Organic Process Research and Development 2009, 13(2):285-291) on the basis of decreasing risk potential, as:
Class 1—Impurities known to be genotoxic and carcinogenic
Class 2—Impurities known to be genetoxic
Class 3—Alerting structure, unrelated to API and of unknown genotoxic potential
Class 4—Alerting structure related to the API
Class 5—No alerting structure
Solvents used in the last step of the API manufacturing process, in particular for the preparation of the desired API salt, its crystallization and any other kind of API purification must be selected taking into account the properties of both solvents and substrates. The used solvents should be inert. For inert solvents are generally meant those which do not react with the salts of API, with API and/or with the salt forming acid or base.
Although crystallisation from a solvent is an important tool to remove impurities from a salt of an API, when inappropriate solvent is used, the crystallized salt could be contaminated by new impurities.
The European Medicine Agency (EMEA) has divided organic solvents usually employed in API manufacturing processes in three categories on the basis of their toxicological properties (EMEA: Notice for Guidance on Impurities: Residual Solvents, CPMP/ICH/283/95, March 1998).
Solvents of EMEA class 3 (acetone, 2-propanol, ethyl acetate etc.) are the preferred ones for the preparation and purification of salts of basic API with acids, on condition that they do not react with the active base substrate, the acid and/or the salt. Indeed, certain precautions must be taken when the basic API and or the acid contain chemical groups that can react with the solvent.
As a general rule, use of alkyl esters as solvents should be avoided, when API is a primary or secondary amine, as the amino group is reactive with respect to the carboxyalkyl moiety yielding amides. This would lead to partial loss of API and generation of API structurally related impurities (March's Advanced Organic Chemistry, Reactions, Mechanism, and Structure 6th Edition, Michael B. Smith and Jerry March, John Wiley & Sons, Inc. Hoboken, N.J., 2007, 1434-1436).
Moreover, alkylesters, such as ethyl acetate, might react with alkyl and aryl sulfonic acids, used for the salification of the active substance free base, with formation of an alkylester, e.g. the ethyl ester, of the sulfonic acid of choice.
With APIs containing primary and/or secondary amino group(s), use of ketones as solvents is generally not advisable as they could condensate with primary amines (Schiff bases formation) and secondary amines (enamines formation) generating API structurally related impurities (March's Advanced Organic Chemistry, Reactions, Mechanism, and Structure Sixth Edition, Michael B. Smith and Jerry March, John Wiley & Sons, Inc. Hoboken, N.J., 2007, 1281-1284).
In addition, ketones in the presence of both strong acids and bases could promote formation of impurities due to self condensation.
Use of alkanols as solvents should be avoided as primary, secondary and tertiary alcohols can react with the acid of choice generating the respective alkylester, which might exhibit alkylating ability and, consequently, might have genotoxic effects (L. Miller et al., Regulatory Toxicology and Pharmacology 44 (2006), 198-211).
The effect becomes more evident when the acid of choice is a strong acid and it is used in a quantity which exceeds the stoichiometic amount and/or the base is added to the acid. Mesylate esters of lower (C1-C5) alkanols, in particular (C1-C3) alkanols, are reactive, direct-acting, substances which have revealed that their DNA alkylation action can induce mutagenic, carcinogenic and teratogenic effect.
Because of their ability to induce genetic mutations and/or chromosomal rearrangements, control of genotoxic and carcinogenic impurities in pharmaceutical substances has become relevant in recent years.
Guidelines from Regulatory Agencies have been recently published outlining limits for testing and control of potential genotoxic impurities (GTIs). For example the European Medicines Agency (EMEA) Committee for Medicinal Products for Human Use (CHMP) has defined a threshold of toxicological concern (TTC) according to which a 1.5 μg/day intake of a genotoxic impurity is considered to be an acceptable risk. From this threshold value, a permitted level in the active pharmaceutical ingredient (API) can be calculated based on the expected daily dose of the active ingredient [Concentration limit (ppm)=TTC [μg/day]/dose (g/day)]. For a drug taken at a dose of 100 mg/day, this equates to a concentration limit of 15 ppm for each potential genotoxic impurity. This represents levels significantly lower than the conventional ICH (International Conference on Harmonization) (ICH Harmonized Tripartite Guidelines, “Impurities in new drug substances” Q3A(R2), 25 Oct. 2006) identification threshold for impurities, which provides a challenge for analytical scientist to develop sensitive analytical methodologies for monitor and quantify the GTIs.
Concerns which are emerging over the possible formation of sulfonic acids lower alkyl esters during the preparation of sulfonate salts (Elder D. P. et al., J. Pharmacy and Pharmacology, 2009, 61:269-278) of API by addition of the alkyl or aryl sulfonic acid (e.g., methanesulfonic acid) to the free base dissolved in the lower alkanol solvent, have led Regulatory Agencies to require applicants for marketing authorization to demonstrate that the drug has a content of alkyl and aryl sulfonates that do not exceed the limits indicated by the Authorities. (Lutz Muller et al., Regulatory Toxicology and Pharmacology 2006, 44; 198-211). Thus it becomes mandatory to verify that formation of alkyl- or aryl-sulfonic acid ester does not occur during the preparation of the API salt and assure that commercial alkyl or aryl sulfonic acids, in particular methanesulfonic acid, used as acid starting materials are not contaminated by the corresponding lower alkylesters. EMEA guidelines for genotoxic impurities set limits for methyl, ethyl, isopropyl methanesulfonates (MMS, EMS, IMS), besylates and tosylates (EMEA/CHMP/CVMP/QWP/66297/2008 of Jan. 24, 2008 and EMEA/CHMP/QWP/251334/2006).
In this framework, Regulatory Authorities (EDA, EMEA) continue to request developing processes for obtaining active substances with higher purity degree and lowest environmental impact.
Testing for genotoxic impurities in active pharmaceutical ingredients (API) involves a number of challenges common to trace analysis.
The most significant analytical challenges are related to three main problems: the first one regards the structural difference between the genotoxic impurities and the main compound, so that different analytical approaches are needed; the second one is related to the respective reactivity or instability, so that special handling techniques are required; the third one is introduced by the sample matrix where “matrix” means all components but analite, i.e. sample solubility and/or chromatographic interferences due to the main components.
Even if analytical methods for the most common genotoxic impurities are described in the relevant literature, each new sample matrix needs to be studied for optimizing analytical selectivity and sensitivity.
The first step of the development of an analytical method for quantification of genotoxic impurities is the selection of the analytical technique; this choice is based on the chemical structure of the analyte and on the limits to be determined. Commonly used techniques are gas chromatography for the volatile genotoxic impurities and HPLC for the non-volatile ones. The use of the mass detector in Single Ion Monitoring (SIM) is considered as the most versatile, sensitive and selective technique for trace analysis, but the type of instrument available, especially the ionization source, and the analysts expertise are critical issues. Flame Ionization Detector (FID), Electron Capture Detector (ECD) and Ultraviolet UV detection can also be used if separation of the analytes peak from the API peak is sufficient; however these methods are less selective.
For very reactive and unstable compounds the derivatization approach can be considered: however, this approach cannot be used if the derivatizing agent can react with the API itself. In this case, the matrix deactivation or elimination or the direct analysis have to be optimized.
Finally, validation issues should be considered. Methods used for the control of genotoxic impurities can be based on limit tests or quantitative tests. In the first case the analyte in the tested sample is compared with a standard solution containing it in a known concentration and the evaluation is based on the determination whether the analyte response is lower or higher than the standard response, in the second case the concentration of the analyte is numerically defined. The extension of the validation depends on the evaluation method, which is chosen, being the requirements, of the validation for a quantitative method more stringent than those for a limit method: specificity as no interference assessment and sensitivity as demonstration that the Limit of Detection (LOD) is lower than the required limit have to be demonstrated by using the limit test approach, while linearity and Limit of Quantitation (LOQ), precision, accuracy and robustness are also necessary by using the quantitative approach.
According to the above mentioned guidelines Q3A(R2) of 25 Oct. 2006, impurities contained in an amount of 0.10% or above in new drug substances (API) to be administered at a daily dose lower than 2 g/day, should be identified (i.e. their structural characterisation has to be achieved); moreover; impurities contained in an amount of 0.15% or above should be qualified (i.e., biological data establishing safety at the specified level should be achieved).
In order to decrease the risks due to the use of solvents in the synthesis stage of an API, efforts have been devoted to the aim of running reactions in the absence of organic solvents. However, often, the advantage of solvent-free liquid phase reactions is decreased by the fact that the use of organic solvents may be requested in the final purification steps, (Koichi Tanaba, Solvent-free Organic Synthesis, 2009 Wiley-VCH).
On the other hand, notwistanding the use of organic solvents in the formation or crystallization of solid state API salts is quite common in the pharmaceutical practice, it may involve environmental problems, such as the risk of danger of fire and explosion, and the toxicity against the workers, in addition to the problems which may arise from contamination of the finished medicament by residual solvents. The residual amounts of the solvent(s) in the active ingredient and/or in the finished medicament can be decreased only by an extension of the drying time or a prolonged heating of the API solid state salt and/or the finished pharmaceutical form, leading to a disadvantageous decrease in the productivity of the whole manufacturing process.
As a matter of fact, when organic solvents are employed for the preparation and/or crystal ligation of API salts, such as ralfinamide methanesulfonate or its R-enantiomer, these salts are contaminated by a residual amount of organic solvents. In the case of formation or crystallization of said methanesulfonate salts from either lower alkanols or alkylesters, formation of lower alkyl esters of methanesulfonic acid may occur in the final product and said impurities may be present as genotoxic contaminants. Moreover, when the residual solvent is either a lower alkanol or alkylester, a lower alkylester of methanesulfonic acid might be formed.